ABSTRACT Breast cancers that are "triple-negative" for the clinical markers ESR1, PGR, and HER2 typically belong to the Basal-like molecular subtype. Defective Rb, p53, and Brca1 pathways are each associated with triple-negative and Basal-like subtypes. Our mouse genetic studies demonstrate that the combined inactivation of Rb and p53 pathways is sufficient to suppress the physiological cell death of mammary involution. Furthermore, concomitant inactivation of all three pathways in mammary epithelium has an additive effect on tumor latency and predisposes highly penetrant, metastatic adenocarcinomas. The tumors are poorly differentiated and have histologic features that are common among human Brca1-mutated tumors, including heterogeneous morphology, metaplasia, and necrosis. Gene expression analyses demonstrate that the tumors share attributes of both Basal-like and Claudin-low signatures, two molecular subtypes encompassed by the broader, triple-negative class defined by clinical markers.

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To report on a highly unusual case of a 20-year-old woman who presented with multifocal metaplastic breast cancer and was subsequently found to carry deleterious germline mutations in both BRCA1 and p53. Genetic testing was requested on an expedited basis to assist in surgical decision-making and BRCA1/2 and p53 genetic analysis was ordered concurrently. BRCA1/2 and p53 analyses were completed using a combination of direct DNA sequencing and multiplex ligation probe amplification (MLPA). The patient was found to carry a deletion of exon 3 of the BRCA1 gene and a splice site mutation at the exon4/intron4 boundary of the p53 gene. To our knowledge, this is the first report of double heterozygosity in BRCA1 and p53. The patient's clinical presentation is highly reminiscent of that predicted by preclinical mouse models. In patients with early onset breast cancer, the possibility of germline mutations in more than one cancer susceptibility gene should be considered. This could have important clinical implications for patients and their at-risk family members.

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Cooperativity of Rb, Brca1, and p53 in Malignant BreastCancer EvolutionPrashant Kumar1, Malini Mukherjee2, Jacob P. S. Johnson1, Milan Patel1, Bing Huey3,Donna G. Albertson3, Karl Simin1*1Department of Cancer Biology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America, 2Department of Pediatric Hematology/Oncology, Texas Children’s Cancer Center, Baylor College of Medicine, Houston, Texas, United States of America, 3Helen Diller Family Comprehensive Cancer Center,University of California San Francisco, San Francisco, California, United States of AmericaAbstractBreast cancers that are ‘‘triple-negative’’ for the clinical markers ESR1, PGR, and HER2 typically belong to the Basal-likemolecular subtype. Defective Rb, p53, and Brca1 pathways are each associated with triple-negative and Basal-like subtypes.Our mouse genetic studies demonstrate that the combined inactivation of Rb and p53 pathways is sufficient to suppressthe physiological cell death of mammary involution. Furthermore, concomitant inactivation of all three pathways inmammary epithelium has an additive effect on tumor latency and predisposes highly penetrant, metastaticadenocarcinomas. The tumors are poorly differentiated and have histologic features that are common among humanBrca1-mutated tumors, including heterogeneous morphology, metaplasia, and necrosis. Gene expression analysesdemonstrate that the tumors share attributes of both Basal-like and Claudin-low signatures, two molecular subtypesencompassed by the broader, triple-negative class defined by clinical markers.Citation: Kumar P, Mukherjee M, Johnson JPS, Patel M, Huey B, et al. (2012) Cooperativity of Rb, Brca1, and p53 in Malignant Breast Cancer Evolution. PLoSGenet 8(11): e1003027. doi:10.1371/journal.pgen.1003027Editor: Sharon E. Plon, Baylor College of Medicine, United States of AmericaReceived February 10, 2012; Accepted August 23, 2012; Published November 15, 2012Copyright: ? 2012 Kumar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Funding: This work was supported by ACS IRG-93-033-15, the Simeon J. Fortin Charitable Foundation, charitable contributions by N. Ekross and Dr. S. Gupta toKS, and NIH CA84118 to DGA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Competing Interests: The authors have declared that no competing interests exist.* E-mail: karl.simin@umassmed.eduIntroductionThe dire need for more effective treatments for aggressive breastcancers has motivated intensive investigations into their cellularand molecular etiology. Breast cancers classified as ‘‘triple-negative’’ by clinical diagnostic markers (ESR1, PGR, andHER2 negative) are heterogeneous in their clinical behavior,morphology, and molecular biology. Triple-negative breastcancers (TNBC) typically express the Basal-like molecularsignature, thus TNBC and Basal cancer classifications arefrequently used interchangeably. However, they are not com-pletely synonymous [1,2]. TNBCs also include the Claudin-lowmolecular subtype [3], which is characterized by greatly reducedexpression of intercellular junction components and by activationof molecular pathways associated with epithelial-to-mesenchymaltransition (EMT), cancer stem cells, and the immune response [4].Histologically,mosttriple-negativebreastcancersareinvasiveductalcarcinomas, but TNBCs also include the metaplastic, medullary, andadenocystic histologic special types, distinctive morphologies that areprevalent among Claudin-low tumors [4]. TNBCs are insensitive toendocrine therapy and HER2 antagonists, but they are sensitive tochemotherapy. Nevertheless, long-term patient outcomes are poor dueto high rates of relapse and acquired chemoresistance [5,6]. MousemodelsthatmimicthecomplexityofTNBCwillbeinvaluabletoolsfordefining the diverse cellular biology and behavior of these tumors andfor rigorously triaging new drug candidates.Basal-like breast cancers often simultaneously inactivate threetumor suppressors that are infamous for their roles in familialcancers: Rb (RB1) [7], p53 (TP53) [8], and BRCA1 [9]. p53 ismutated in 20–30% of human breast cancers and defectivepathway intermediates also increase breast cancer risk [10].Moreover, nearly all Basal-like cancers with BRCA1 mutation haveconcomitant p53 mutation [11]. Germline BRCA1 mutationpredisposes early-onset breast cancers that are often triple-negativeand that correlate with Basal-like tumors in microarray analyses[9]. BRCA1 mutation accounts for nearly half of familial breastcancers (OMIM 604370), but BRCA1 is also down-regulated insporadic breast tumors without germline mutation [12]. Theoverall effect of BRCA1 loss is likely pleiotropic. The best studiedfunction of BRCA1 is in orchestrating DNA double-strand breakrepair through homologous recombination or non-homologousend-joining. The importance of defective DNA repair in breastcancer is underscored by the observation that all known genesassociated with inherited forms of the disease safeguard genomicintegrity [13]. BRCA1 also functions as a transcription factor [14]that appears to be required for the differentiation of stem/progenitor cells into mature luminal cells [15]. This finding isconsistent with the recent characterization of BRCA1-associatedtumors as aberrant luminal progenitor cells [16].The pair-wise cooperativity of Brca1 and p53 in mammarytumorigenesis has been studied in mouse models [17]. We andothers have investigated the impact of combined Rb and p53inactivation on mammary tumorigenesis in vivo [18–20]. Weshowed that following Rb perturbation in vivo, tumor progression islimited largely by p53-dependent apoptosis, and that loss of thesecond p53 allele in MFT121+/p53f/+tumors is likely a prerequisitePLOS Genetics | www.plosgenetics.org 1November 2012 | Volume 8 | Issue 11 | e1003027

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for mammary tumor progression [19], since the vast majority oftumors lose the wild type p53 allele during tumorigenesis. In thecontext of a brain carcinoma model initiated by T121, apoptosisappears to be the critical function of the normal p53 allele that isthe target of selective pressure [21].Our motivation to combine Rb inactivation with Brca1 and p53mutation derives, in part, from the observation that the Rb gene isamong the most frequently deleted loci in Brca1/p53-mutatedmouse tumors [22], indicating that Rb is a critical barrier to tumorprogression. Rb pathway inactivation is strongly associated withhuman triple negative breast cancers. A cardinal feature of basal-like breast cancers is the abundant expression of the ‘‘proliferationcluster’’ genes [7], which include many E2F-regulated genes thatare de-repressed following Rb inactivation. In human breastcancers, reduced pRb activity correlates with higher tumor grade[23], but also predicts improved chemotherapy responsiveness [24].The Rb gene itself is mutated in breast cancer [25], and recentgenomic studies have indicated an overrepresentation of mutationswithin pRb-binding sites of human gene regulatory domains [26].In this study we show that mammary tumors caused byinactivation of the pRb family (pRbf) of proteins (pRb, p107, p130),together with Brca1 and p53 inactivation, mimic several aspects ofthe most aggressive forms of breast cancer, including rapid tumorprogression, poor differentiation, distant metastasis, necrosis,metaplasia, and genomic instability. Our findings illustrate thecompounding effect of acquiring multiple tumor suppressormutations during tumor evolution and underscore the distinctrequirements of each of these canonical tumor suppressor proteins.ResultsConditional T121expression in mammary epitheliumWe constructed the MFT121(MMTV-Floxed-eGFP-T121) trans-gene to conditionally inactivate the pRb family (pRbf) of pocketproteinsinmammaryepithelium(Figure1A).TheT121proteinisanamino-terminal fragment of the SV40 large T antigen that perturbspRbfactivity and predisposes tumorigenesis in a range of tissues[27]. While Rb inactivation alone is sufficient to induce mammarytumors [20], the shorter latency of TgWAP-T121tumors indicatesthere is functional redundancy or compensation by the relatedpocket proteins p107 or p130 [19]. In the MFT121model, theMMTV-LTR promotes mammary-specific transgene expression(Figure 1B). The approach of inactivating pRBf, Brca1, and p53specifically in mammary epithelial cells via the Wap-Cre transgene[28] enabled us to avoid the appearance of lymphomas [20] orsarcomas [29]. Wap-Cre excised the LoxP-eGFP-stop-LoxPreporter cassette and initiated T121 expression in ductal andalveolar luminal epithelial cells (Figure 1C). Virgin glands ofMFT121;WAP-Cre miceappeared normal.Lactatingglands (WAP-Cre-induced) showed reduced alveolar density, similar to the glandatrophy phenotype in the TgWap-T121model, which was associatedwith apoptosis caused by T121-induced proliferation [19] andlactation defects observed in WAP-Cre; Rbfl/fl; p1072/2mice [20].Concomitant pRbf, p53, and Brca1 inactivationsignificantly accelerates tumor onsetMultiparous TgMFT121; TgWap-Cre mice remained tumor-freefor more than a year after Cre-induction, but mice that were eitherFigure 1. The MFT121 transgene construct. (A) The MFT121transgene construct. The ‘‘floxed’’ eGFP-stop cassette was expressedthroughout virgin mammary epithelium (B, original magnification 506).Following Cre-induced excision, T121was expressed (red) in the majorityof luminal epithelial cells (C). eGFP immunolabeling (green) revealednon-recombined cells (original mag. 4006). Kaplan-Meier analysis oftumor onset (D). p53 was haploinsufficient (dashed green) for tumorsuppression (p,0.0001, log-rank test). Homozygous p53 mutation (solidgreen) shortened tumor latency. Brca1 loss (solid red) furtheraccelerated tumor onset (p,0.0005). Median tumor latency of TBPmice was approximately seven weeks. Parturition Day 1=Time 0. Allmice harbored the Wap-Cre transgene (not shown). Significance levelsfor critical comparisons are indicated.doi:10.1371/journal.pgen.1003027.g001Author SummaryThese studies establish a unique animal model ofaggressive forms of breast cancer for which there are noeffective, targeted treatments. Rb, p53, and Brca1 areassociated with inherited forms of cancer, but defects inthese pathways are also found together in a subset ofbreast cancer patients without a family history of thedisease. Simultaneous inactivation of all three pathwayscauses more aggressive disease than do pair-wise combi-nations, indicating that the pathways play non-overlap-ping roles in tumor prevention.Genetic Interaction of pRb, Brca1, and p53PLOS Genetics | www.plosgenetics.org2 November 2012 | Volume 8 | Issue 11 | e1003027

others that Rb/p53 tumors can also resemble TNBC and theClaudin-low molecular phenotype [3,18,20]. A single TP tumorclustered among the previously designated Group II tumors(Figure 6A, yellow box), which are the paradigm cases of theClaudin-low subtype [3]. In addition, a single TP tumor clusteredwith tumors with a squamous metaplastic histology. Finally, twoTP tumors co-segregated with the TBP tumors (Figure 6A, orangebox), which is not surprising given that Rb is one of the mostfrequently deleted loci among Brca1/p53-mutated mouse tumors[22].Pathway analysisThe similarity between TBP tumors and human Claudin-lowand Basal-like cancers was also evident from pathway analysis ofup-regulated genes of each of the three tumor types (Figure 7A,File S1). We queried the KEGG (Kyoto Encyclopedia of Genesand Genomes) and GO (Gene Ontology) databases with lists ofgenes that were differentially expressed by TBP tumors (seeMethods) and by human Claudin-low and Basal-like tumors [4].Cytokine, chemokine, and MAPK signaling pathways rankedhighly among both Claudin-low and TBP tumors. Pathways thatare enriched in cancers of diverse origins ranked highly in bothBasal-like and murine TBP tumors.The GO terms associated with the respective tumor types wereconsistent with the enriched KEGGpathways. Cell-cycle progression(GO:0007049, p=2.43551E-59) and DNA repair (GO:0034984,p=6.95081E-22) dominate the list of functions enriched in Basal-liketumors (File S1). Similarly, regulation of cell proliferation(GO:0042127, p=6.01E-13) is among the top terms for TBPtumors. The three top scoring, inter-related GO terms for TBPtumors are regulation of developmental process (GO:0050793,p=7.50E-16), organ morphogenesis (GO:0009887, p=3.53E-14),and tissue development (GO:0009888, p=1.36E-13). These GOterms are reflective of the enrichment of the Wnt, ErbB, TGF-b, andVEGF signaling pathways identified by KEGG pathway analysis.Claudin-low tumors are enriched for wound (GO:0009611,p=4.29939E-66)andinflammatoryp=1.26817E-50), which are also among the top functions associatedwith TBP tumors (7.37E-13 and 6.46E-12, respectively).responses(GO:0006954,CGH analysisGiven the requirement for BRCA1 in DNA damage repair andcentrosome regulation, we tested the hypothesis that TBP tumorsharbor more genomic copy number aberrations (CNAs) than doTP tumors with intact Brca1. We enumerated CNAs by counting‘‘copy number transitions,’’ the number of changes in the CGHprofile from one copy number level to another that occur withinchromosomes [34]. Unexpectedly, we found no statisticallysignificant difference (p=0.8374) in the mean number of CNAsbetween TBP tumors (n=8) and TP tumors (n=10) using array-based comparative genomic hybridization (aCGH).The low multiplicity of TBP and TP tumors (1–3 per mouse)and their latency indicate that combinations of pRbf, Brca1, andp53 pathway perturbations are not sufficient for malignanttransformation in our models. To identify potentially collaboratingoncogenic events, we manually curated loci with copy numberchanges (see Methods). In nine tumors (50%), we observedrecurrent losses of large, variable regions spanning chr4 and chr10(Figure 8). Both chromosomes harbor many potential tumorsuppressors, including regulators of cell death, such as Tm2d1,Utp11l, Trp73, Dffa, Runx3, Lck, Dhcr24, Faf1, Pax7, and Casp9, andeffectors of cell death, such as Col18a1, Gadd45b, Dapk3, andCasp14. Among all the tumors assayed (n=18), we identifiednearly five-hundred loci (Table S9 in File S1) with potential copynumber gains. Approximately half of the genes are included oncurated lists of cancer-associated genes, including the CancerGene Census (Sanger Institute) and the KEGG Pathways inCancer. We observed focal amplification of several canonicalproto-oncogenes, including c-Myc amplification (log2ratio=3.64,p,0.0001) in a single TP tumor, H-ras amplification in two of tenTP tumors, and K-ras amplification in two of eight TBP tumors.Pathway analysis of these five-hundred putative collaboratinggenes revealed enrichment of several signaling pathways, includingthe MAP Kinase, Focal Adhesion, Wnt, and ErbB pathways(Table S10 in File S1).DiscussionHere we report a highly penetrant engineered mouse model ofTNBC. Our previous work showed that when pRbfand p53 aresimultaneously perturbed in mammary epithelium, adenocarcino-mas develop with long latency, suggesting a requirement foradditional oncogenic events. However, these mouse tumorsdisplayed only limited chromosomal copy number aberrations[19]. Because genomic instability is a hallmark of malignanttransformation [35], especially among BRCA1 familial cancers[36] and aggressive sporadic breast cancers [37], we hypothesizedthat Brca1 mutation would accelerate the tumor development weobserved following dual inactivation of pRbfand p53. Our resultsshow that concomitant inactivation of all three tumor suppressorpathways in mammary epithelium has an additive effect on tumorlatency and predisposes highly penetrant, malignant carcinomas.Although Brca1 inactivation accelerated tumorigenesis comparedto TP tumors, we observed no statistical difference betweenchromosomal copy number transitions in tumors with or withoutBrca1, despite the extensive CNAs observed by others in Brca1/p53 tumors [22].The pRb-regulated cell cycle network is frequently disrupted inTNBC tumors [7,26,37,38], and the Rb locus is among the mostfrequently lost in Brca1/p53 mouse tumors [22], indicating thatthere is strong selective pressure for Rb pathway inactivation. Wespeculate that direct inactivation of pRbfby T121may allow TBPcells to escape this rate-limiting barrier of transformation withoutaccruing numerous chromosomal aberrations. Thus, in thecontext of defective pRb and p53 function, tumor progressionmay be unrelated to the proportion of the genome altered by copynumber alterations. It will be important to determine the effect ofBrca1 loss on the abundance and identities of somatic mutationsthat are not detectable by CGH.The importance of p53 mutation in breast cancers is welldocumented and is confirmed in the present study. The dualinactivation of pRbf and Brca1 caused markedly increased celldeath that was reduced by p53 mutation. p53-independent cell deathlikely remains a significant barrier to tumor progression amongTBP tumors and may account, in part, for the observed loss ofgenomic regions that harbor cell death regulatory genes, mostnotably on chr4 where the p53 paralog p73 resides. Identifying thegenomic alterations that are conserved across species will be usefulstaining (K). Abundant E-cadherin (CDH1, green) in normal adjacent (H) or well-differentiated tumor (I). Reduced or absent CDH1 along invasivetumor fronts (J–L). Keratinic whorls in squamous metaplastic cells (M, asterisks). Whorl-associated and disseminated Keratin 6 expression (N, green).Pulmonary metastases were observed in both T121/p53 and TBP mice (O, arrow).doi:10.1371/journal.pgen.1003027.g004Genetic Interaction of pRb, Brca1, and p53PLOS Genetics | www.plosgenetics.org 7November 2012 | Volume 8 | Issue 11 | e1003027

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in evaluating the impact of the myriad of CNAs observed in breastcancers and may help to explain the heterogeneity of TNBCs.Brca1 mutation not only accelerated tumor development butalso shifted the tumor spectrum. Whereas T121/p53 mouse tumorsoften resembled the Luminal-B molecular subtype breast cancers,which show relatively abundant expression of luminal epithelialcell differentiation markers, TBP tumors consistently sharedfeatures of Basal-like and Claudin-low molecular subtypes. Othershave argued that Basal-like and Claudin-low gene expressionsignatures reflect progenitor and stem cell phenotypes, respectively[4,16], consistent with a role for Brca1 in mediating stem/progenitor cell maturation [15]. Loss of BRCA1 activity may alsoalter tumor phenotype through deregulation of the EMT inducerSLUG [39].The CGH analysis of our mouse tumors revealed CNAsconsistent with mutations observed in genomic surveys of humanbreast cancers [40,41]. Similar to the studies of human tumors, wesaw increased copy numbers of known oncogenic driver genes,including myc, egfr, crebbp, jak1, H-ras, and K-ras, as well asenrichment of pathways implicated in tumor progression, includ-ing the WNT signaling pathway, regulation of actin cytoskeleton,focal adhesion, cell shape, and mobility proteins. Far fewerinvestigations have focused on genetic deletions and cancerdevelopment mechanisms. We also found decreased copy numbersof known tumor suppressors, including map2k, ppp2r, and pten.Given the strong similarities between our mouse model andaggressive human breast cancers, the TBP model provides aninvaluable preclinical platform to identify and assess potentialtherapeutics for aggressive and chemoresistant breast cancersubtypes [42,43].Materials and MethodsEthics statementThis study was performed in strict accordance with therecommendations in the Guide for the Care and Use ofLaboratory Animals of the National Institutes of Health.Derivation of MFT121transgenic miceThe LoxP-eGFP-Stop-LoxP cassette and T121-encoding DNAwereclonedintoEcoRIHindIIIsitesofMMTV-SV40-Bssk(Addgeneplasmid 1824). The LoxP-eGFP-Stop-LoxP cassette was providedcourtesy of the T. Jacks lab. Resulting and subsequent generationMFT121transgenic mice were identified by PCR amplification of a215-bp fragment using the oligo pair: 59-GCATCCAGAAGCCTC-CAAAG -39 and 59-GAATCTTTGCAGCTAATGGACC-39 com-plementary to the T121sequence. Cre transgenic mice were identifiedusing the oligo pair: 59-TGATGAGGTTCGCAAGAACC-39 and59-CCATGAGTGAACGAACCTGG-39. The cycling profile was94uC for 2 min., 35 cycles of 94uC for 20 sec., 62uC for 45 sec., and72uC for 45 sec.; the final incubation of 72uC was for 2 min. Weestablished five TgMFT121founder transgenic lines, though three linesfailed to express the eGFP reporter. We describe here our studies ofthesinglemouselinewithhighereGFPexpressioninvirginmammaryglands. eGFP expression was also evident in salivary glands and footpads in this line (data not shown).Figure 5. Cross-species comparison of breast cancers. TBP (blackboxes, n=8) and TP (open black boxes, n=9) tumors were compared topublished mouse (gray boxes, n=135) and human (n=337) microarrayexpression profiles (colored according to PAM50 subtype). Most (76%)of our TgMFT121mouse tumors cluster with human Basal-like breastcancers (red boxes). The Treeview files of the clustering analysis areavailable in File S2.doi:10.1371/journal.pgen.1003027.g005Genetic Interaction of pRb, Brca1, and p53PLOS Genetics | www.plosgenetics.org 8November 2012 | Volume 8 | Issue 11 | e1003027

RNA was purified using the Qiagen RNeasy Mini Kit according tothe manufacturer’s protocol using 20–30 mg tissue. RNA integritywas assessed using the RNA 6000 Nano LabChip by Bioanalyzer(Agilent). Two micrograms of total RNA were reverse transcribed,amplified, and labeled with Cy5 using a Low RNA InputAmplification kit (Agilent). Common reference RNA consisted oftotal RNA harvested from equal numbers of C57Bl6/J and 129maleand femaleDay1pups(courtesyofDr.CamPatterson,UNC).Reference RNA was reverse transcribed, amplified, and labeledwith Cy3. The amplified sample and reference were co-hybridizedovernight to Agilent Mouse Oligo Microarrays (G4121A). Theywere then washed and scanned on an Axon GenePix 4000Bscanner, analyzed using GenePix 4.1 software, and uploaded intothe UMD database (https://genome.unc.edu/) where Lowessnormalization is automatically performed. All data were submittedto GEO (GSE34479). The genes for all analyses were filtered by: 1)requiring intensity values in both channels to have a mean Lowessnormalized intensity of .10, 2) Values being reported in .70% ofthe samples, and 3) the absolute value of the log2of the ratio ofChannel 2/Channel 1 for at least three arrays having to be .1.6.Hierarchical clustering was performed using Cluster v3.0 anddisplayed using JavaTreeview v1.0.8.We identified 871 differentially expressed TBP transcripts usingSAM implemented in BRB-ArrayTools (R. Simon and the BRB-ArrayTools Development Team, NCI; Table 1; FDR 0.0485,delta 0.92931). Gene ontology analyses were performed using theFatiGO tool (Babelomics ver. 4.2, babelomics.bioinfo.cipf.es).Mouse gene symbols were converted to human EntrezIDs usingAgilent annotations and the Mouse Genome Informatics databaseof The Jackson Laboratory for comparisons. The Fisher’s exactFigure 8. Comparative genomic hybridization. TBP tumors (n=8) and TP tumors (n=10) were analyzed by array CGH to identify copy numberaberrations (CNAs). Green lines to the right of the chromosome ideograms indicate gains and red lines to the left indicate losses of individual tumorsamples. No significant difference was found between the average number of CNAs between TBP and TP tumors (p=0.8374). Frequent losses wereseen on chromosomes 4 and 10. Other recurrent losses included chromosome 7. Frequent gains were associated with chromosome 6.doi:10.1371/journal.pgen.1003027.g008Genetic Interaction of pRb, Brca1, and p53PLOS Genetics | www.plosgenetics.org 11 November 2012 | Volume 8 | Issue 11 | e1003027